Project Summary Human Immunodeficiency Virus 1 (HIV-1) has evolved sophisticated strategies to limit the presentation of viral epitopes to the immune system. This is evidenced by the ability of the virus to minimize the number of exposed Envelope proteins on the virus surface at the expense of virus infectivity. How HIV-1 regulates the incorporation of viral Envelope proteins into assembling particles is still very unclear. We hypothesize that specific motifs found within the HIV-1 Envelope cytoplasmic domain as well as host cell membrane lipids regulate the acquisition of Env into assembling virus particles. In our first aim of this proposal, we will quantify the single molecule nanoscale dynamics of HIV-1 Envelope and mutants of the cytoplasmic domain on the surface of producing cells. In our second aim, we will measure the three-dimensional (3D) structural organization and angular distribution of HIV-1 Envelope on the surface of budding virus particles. For both aims, we propose to identify key viral and host cell membrane determinants responsible for the spatiotemporal association of HIV-1 Envelope with nascent virus assembly sites. Both aims will utilize the tens of nanometer resolution and molecular specificity of three-dimensional superresolution microscopy to illuminate the dynamics and organization of HIV-1 Envelope. Our quantitative approach will provide the necessary foundational knowledge for constructing accurate models of HIV-1 Envelope acquisition into virus assembly sites. We believe that these models will aid in the future development of novel strategies aiming to inhibit accessibility of HIV-1 Envelope to virus assembly sites.